One of the modern strategies for treating autoimmune diseases such as rheumatoid arthritis and psoriasis involves the use of biologic TNF receptor decoys, such as soluble receptors or therapeutic antibodies, to intercept the inflammatory ligand TNF-1, and thus block the pathological activation of its receptors. However, current TNF-1 biologic blockers are all dimeric in structure, whereas TNF-1 itself is homotrimeric in nature. From a structural biology point of view, a homodimeric structure with a two-fold symmetry cannot perfectly dock to a homotrimeric structure with a three-fold symmetry, thus limiting the affinity between the two molecules. Here we describe a general methodology for efficient creation of trimeric soluble receptors as secreted proteins. The process involves gene fusion between a soluble receptor with a ligand binding domain or any biologically active protein and a trimerization tag from the C-propeptide domain of pro-collagen (Trimer-Tag), which is capable of self-assembly into a disulfide bond-linked trimer. We show that the homotrimeric soluble TNF receptor produced with such method is a more potent blocker than dimeric TNF receptor decoys in inhibiting TNF- 1 signaling in vitro. Moreover, we have also demonstrated that covalently strengthened homotrimeric TRAIL/Apo2L-Trimer ligand is a potent anticancer agent, in contrast to its dimeric Fc fusion counterpart. Thus, Trimer-Tag has the potential to become a new platform technology for rational design of the next generation biologic drugs against autoimmune diseases, cancer, AIDS, osteoporosis, and heart disease. In this Phase I SBIR application, we seek to significantly increase the expression level and optimize the purification scheme of these recombinant trimeric fusion proteins in the hope that this novel technology, which is covered by 3 U.S. patents, can quickly move from preclinical stage towards the bedsides of millions of patients. PUBLIC HEALTH RELEVANCE: This Phase I SBIR application seeks to further optimize and streamline a newly patented protein trimerization technology for the design and production of secreted therapeutic biologics targeting major diseases such as autoimmune diseases, cancer, AIDS, osteoporosis, and heart disease.